Peter M.J.M. Cruijsen

Dynamics of background adaptation in Xenopus laevis: Role of catecholamines and melanophore-stimulating hormone

The pars intermedia of the pituitary gland in Xenopus laevis secretes alpha-melanophore-stimulating hormone (alpha-MSH), which causes dispersion of pigment in dermal melanophores in animals on a black background. In the present study we have determined plasma levels of alpha-MSH in animals undergoing adaptation to white and black backgrounds. Plasma values of black-adapted animals were high and decreased rapidly after transfer to a white background, as did the degree of pigment dispersion in dermal melanophores. Plasma MSH values of white-adapted animals were below the detection limit of our radioimmunoassay. Transfer of white animals to a black background resulted in complete dispersion of melanophore pigment within a few hours, but plasma MSH levels remained low for at least 24 hr. This discrepancy between plasma MSH and degree of pigment dispersion suggested the involvement of an additional factor for stimulating dispersion. Results of in vitro and in vivo experiments with receptor agonists and antagonists indicated that a beta-adrenergic mechanism, functioning at the level of the melanophore, is involved in the stimulation of pigment dispersion during the early stages of background adaptation.

New aspects of signal transduction in the Xenopus laevis melanotrope cell.

Light and temperature stimuli act via various brain centers and neurochemical messengers on the pituitary melanotrope cells of Xenopus laevis to control distinct subcellular activities such as the biosynthesis, processing, and release of alpha-melanophore-stimulating hormone (alphaMSH). The melanotrope signal transduction involves the action of a large repertoire of neurotransmitter and neuropeptide receptors and the second messengers cAMP and Ca(2+). Here we briefly review this signaling mechanism and then present new data on two aspects of this process, viz. the presence of a stimulatory beta-adrenergic receptor acting via cAMP and the egress of cAMP from the melanotrope upon a change of alphaMSH release activity.

Analysis of inositol phosphate metabolism in melanotrope cells of Xenopus laevis in relation to background adaptation

The present study examined inositol phosphate metabolism in melanotrope cells of Xenopus laevis to determine if inositol phosphates are involved in regulating the biosynthetic or secretory activity of these cells. No correlation could be found between inositol phosphate metabolism and the secretory activity of the melanotrope cells. Therefore, we conclude that inositol phosphate production is not directly involved in the regulation of release of alpha-MSH from these cells. However, there were dramatic differences in the capacity of the melanotrope cells to produce inositol phosphates dependent on the state of background adaptation of the animals from which the melanotropes were derived; cells from white-adapted animals had a low capacity to produce inositol phosphates, whereas melanotropes from black-adapted animals had a high capacity in this regard. During adaptation of animals from a white to a black background, the capacity of the melanotrope cells to produce inositol phosphates was only very slowly acquired, reminiscent of the slow acquisition displayed by these cells to produce POMC during background adaptations. Likewise, during black to white background adaptation, the melanotrope cells very slowly lost the capacity to phosphorylate inositol, which correlates with the slow loss of the biosynthetic capacity of melanotrope cells during such adaptations. Altogether we conclude that inositol phospholipid metabolism is likely involved in the regulation of the biosynthetic processes of melanotrope cells of Xenopus laevis.

Intracellular signal transduction by the extracellular calcium-sensing receptor of Xenopus melanotrope cells.

The extracellular calcium-sensing receptor (CaR) is expressed in various types of endocrine pituitary cell, but the intracellular mechanism this G protein-coupled receptor uses in these cells is not known. In the present study we investigated possible intracellular signal transduction pathway(s) utilized by the CaR of the endocrine melanotrope cells in the intermediate pituitary lobe of the South African-clawed toad Xenopus laevis. For this purpose, the effects of various pharmacological agents on CaR-evoked secretion of radiolabeled secretory peptides from cultured melanotrope cells were assessed. CaR-evoked secretion, induced by the potent CaR agonist L-phenylalanine (L-Phe), could not be inhibited by cholera toxin, nor by NPC-15437 and PMA, indicating that neither G(s)/PKA nor G(q)/PKC pathways are involved. However, pertussis toxin (G(i/o) protein inhibitor), genistein (inhibitor of PTKs), wortmannin/LY-294002 (PI3-K inhibitor) and U-0126 (inhibitor of extracellular signal-regulated kinase, ERK) all substantially inhibited CaR-evoked secretion, indicating that the Xenopus melanotrope cell possesses a PI3-K/MAPK system that plays some role in CaR-signaling. Since no direct effect of L-Phe on ERK phosphorylation could be shown it is concluded that CaR must act primarily through another, still unknown, signaling pathway in Xenopus melanotropes. Our results indicate that the PI3-K/MAPK system has a facilitating effect on CaR-induced secretion, possibly by sensitizing the CaR.

Actions of PACAP and VIP on melanotrope cells of Xenopus laevis.

The neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are implicated in the regulation of gene expression and hormone secretion in mammalian melanotrope cells and a mammalian pro-opiomelanocortin (POMC)-producing tumor cell line, but the physiological relevance of this regulation is elusive. The purpose of the present study was to establish if these peptides affect biosynthetic and secretory processes in a well-established physiological model for endocrine cell functioning, the pituitary melanotrope cells of the amphibian Xenopus laevis, which hormonally control the process of skin color adaptation to background illumination. We show that both PACAP and VIP are capable of stimulating the secretory process of the Xenopus melanotrope cell. As the peptides are equipotent, they may exert their actions via a VPAC receptor. Moreover, PACAP stimulated POMC biosynthesis and POMC gene expression. Strong anti-PACAP immunoreactivity was found in the pituitary pars nervosa (PN), suggesting that this neurohemal organ is a source of neurohormonal PACAP action on the melanotropes in the intermediate pituitary. We propose that the PACAP/VIP family of peptides has a physiological function in regulating Xenopus melanotrope cell activity during the process of skin color adaptation.

Intracellular calcium buffering shapes calcium oscillations in Xenopus melanotropes.

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise for the interplay between the burst-like openings of voltage-operated Ca2+ channels and Ca2+-extrusion mechanisms. We have previously shown that Ca2+-extrusion rates increase with increases in [Ca2+]i, suggesting that Ca2+ itself plays a role in shaping the Ca2+ oscillations. The purpose of the present study was to test this hypothesis by manipulating the intracellular Ca2+ buffering capacity of the cell and determining the consequences of such manipulations for the shape of the Ca2+ oscillations. We manipulated the cytosolic buffering capacity by loading the fast Ca2+ chelator BAPTA into cells. During loading the [Ca2+]i was dynamically imaged with confocal laser scanning microscopy. The basal [Ca2+]i was reduced with BAPTA loading and this reduction was associated with lower Ca2+-extrusion rates, a broadening of the Ca2+ oscillations and declined oscillation frequencies. Short loading periods of the buffer led to new, stable patterns of Ca2+ signaling and to reduced but stable levels of peptide secretion. We propose that the cytosolic Ca2+ buffer capacity, and thus by inference the profile of intracellular Ca2+ buffering proteins, is an important factor in setting the frequency and shape of Ca2+ oscillations.